Fluorescence spectroscopy is a widely used technique in the Biological sciences. In fluorescence spectroscopy, light at a specific frequency is absorbed by a given molecule or fluorescent entity (also called fluorophore), exciting its electronic state. The fluorescent entity then emits light at a slightly different frequency, as the fluorophore returns to the original ground state. Fluorescence spectroscopy is analogous to Raman spectroscopy in that a pump light excitation induces the emission of Stokes light, shifted to a lower frequency relative to the pump light. However, fluorescence requires the absorption of the pump light of a specific frequency, the frequency depending on the electronic structure of the fluorescent entity. Also, contrary to Raman scattering, typical Stokes shifts for fluorescence phenomena are a few 10's of nm apart from the pump light, which complicates the cross-talk between the pump light and the Stokes signals at the detection level. Furthermore, as opposed to Raman scattering, which is essentially instantaneous, fluorescence emission takes place across a wide range of lifetimes, within a few ns or up to a few ms, depending on the fluorophore.
Fluorescence spectroscopy methods can be divided into two broad areas: secondary fluorescence and intrinsic fluorescence. Secondary fluorescence uses certain predetermined fluorophores as marker substances (chemical compounds or quantum dots, for example). These fluorophore molecules attach themselves to a specific protein, enzyme, or DNA string, called a target substance; by doing so, their fluorescent capacity is either enhanced or suppressed. The detection of fluorescence activity or its relative change therefore enables the measurement and identification of the desired target. In the past decades, great effort has been devoted to the development of fluorescent molecules that act as chemical markers for a wide variety of target substances relevant in the biochemical, pharmaceutical, and medical arenas. Intrinsic fluorescence uses the fluorescence emission of the target molecules themselves, which limits its application to strongly emitting targets.
Time-resolved fluorescence (TRF) is a technique that has all the advantages of fluorescence spectroscopy, with the added benefit of being intrinsically related to the target substance, eliminating concerns about absolute intensity measurements. As a result, interference from different chromophores, diverse scattering mechanisms from the sample and effects like photo-bleaching become transparent to the technique. This makes TRF a method of choice for developing fluorescence-based sensors for biological and biochemical studies. Typically in these applications, the fluorescent molecules used are large organic complexes or quantum dots such that fluorescent lifetimes are quite short, less than 10 ns. Any application of TRF in this regime implies the use of pulsed lasers and high-end detection techniques: ultra fast photo detectors or high frequency modulators and RF filters.
In general, fluorescence spectroscopy techniques are mostly limited to laboratory environments due to the following reasons:
1) Short lifetime measurement techniques require the use of expensive and delicate equipment: pulsed pump lasers and state-of-the-art synchronized photo-detection schemes.
2) Fluorescence spectroscopy instrumentation is bulky.
3) Conventional fluorescence techniques require the use of high performance optical filters. This adds on to the price of the instrument, its complexity, and reduces the signal collection efficiency. It also increases the cross-talk between pump and Stokes signals, and between the Stokes signals from different fluorophores.
4) Due to the extra complexity and cross-talk added by the optical filtering procedures, only small number of target substances can be analyzed simultaneously (3 or 4 at a time).
5) In fluorescence lifetime measurements, fluorophore concentration values are normally disregarded, as the measurement technique is only involved with relative changes of the signal in time. Also, the analytical complexity of deriving both lifetime and concentration values increases rapidly with the number of targets being analyzed. As a result, current lifetime fluorescent techniques are limited to fixed concentration measurements for a few target substances (2, 3 or 4).
6) Due to the close spectral proximity between the pump and Stokes signals in fluorescence spectra, and between Stokes signals from different fluorophores, high-performance optical filtering techniques are required. This increases cost and complexity of typical fluorescence devices.
In view of the above, there is a need for a TRF device that can be implemented in field applications under harsh environmental conditions. These applications usually require measurement of multiple targets (10 to 25) simultaneously. A complete measurement and sample assessment needs to be performed in a time frame of 1 s or less. Such a device would not only find new applications but also enhance current technologies like DNA sequencing and fluorescence imaging microscopy.